Method of making a vaccine for anthrax

ABSTRACT

A method of making a vaccine for anthracis that inolves a bacterial expression system and production and use of protective antigen (PA) against  Bacillus anthracis . The PA immunogen is useful in a vaccine against human anthrax. The PA can be produced by an asporogenic organism which produces the desired antigen, which is then harvested from the supernatant.

This application is a divisional application of Ser. No. 08/346,238filed Nov. 23, 1994, said application allowed.

FIELD OF THE INVENTION

This invention relates to the bacterial expression system, productionand use of protective antigen (PA) against Bacillus anthracis. The PAimmunogen is useful in vaccine against human anthrax. The PA can beproduced by an asporogenic organism which overproduces the desiredantigen, which is then harvested from the supernatant.

BACKGROUND OF THE INVENTION

Bacillus anthracis is the etiologic agent responsible for anthrax, adisease often found in persons exposed to infected animals or theirproducts. Persons particularly exposed to animals include veterinarians,laboratory technicians, ranchers and employees working with skin or hairof animals. The mode of entry into the body may be the skin or, whencontaminated meat is eaten, the gastrointestinal tract. Inhaling ofspores can cause inhalation anthrax, a disease that can be fatal.Vaccines against Bacillus anthracis have been available. Virulentstrains of the organism produce two toxins and a poly-D-glutamic acidcapsule which are coded for on two endogenous plasmids, pX01 and pX02,respectively. Loss of either of the plasmids results in an attenuatedstrain of reduced virulence, while loss of both results in an avirulentorganism. The history of the USAMRIID Sterne strain of B. anthracisprior to 1981 is uncertain, though it is believed to be derived from theSterne strain isolated at the Onderstpoort Research Laboratory inPretoria, South Africa.

In 1985 the Bacillus anthracis protective antigen (PA) gene was clonedinto a plasmid (pUB110) resulting in the formation of a recombinantplasmid identified as pPA102, which was reported in the literature(Ivins and Welkos, Infection and Immunity, 54:537-542 (1986)). Theproduction of vaccines lacking lethal factor was possible thereby.However, a primary problem remained, since the Bacillus anthracis formedspores. Once spores have formed, they persist in the environment formonths and years. Once the laboratory environment contains such spores,it is very difficult to free the environment of the spores.

It was also previously reported that protective antigen (PA) could beproduced in baculovirus. [Iacono-Connors, et al., Infection andImmunity, 58:366-372 (1990); Iacono-Connors, et al., Infection andImmunity, 59:1961-1965 (1991)] A major problem in production of the PAin the baculovirus disclosed therein is that the desired antigenrequires a complex purification process. Even after purification byimmuno-affinity chromatography, undesired cellular material continues tocontaminate the desired product.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides organisms which produce protectiveantigen (PA) lacking lethal factor and edema factor proteins which, whenpresent as contaminants in vaccine, can cause serious side effects. Theproducing organisms of the invention are also, surprisingly,non-sporulating. Furthermore, the desired antigen is expressed into thesupernatant. Hence, the protective antigen produced is easily purifiedand, though protective, does not cause many of the troublesome sideeffects of prior art vaccines. The organisms of the invention lackingspore-forming function may be killed by heat shock at temperatures aslow as 60° C. for 60 minutes. Hence, contamination of the environmentwith viable spore-forming organisms is easily avoided anddecontamination is easily accomplished.

Genesis of ΔSterne-1(pPA102)CR4:

A 6 kb Bam HI fragment harboring the PA structural gene isolated fromthe endogenous Sterne plasmid pXO1 was ligated into plasmid pBR322 andcloned into Escherichia coli bacteria (Vodkin and Leppla, 1983). Fromthe resultant recombinant plasmid pSE36, the 6 kb fragment was thensubcloned into the gram-positive vector pUB110 using the Bam HIrestriction site. The resulting plasmid was transformed into B. subtilisIS53 and two stable PA producing, kanamycin resistant isolates werefound (pPA101 and pPA102) (Ivins and Welkos, 1986). Subsequent analysisof the plasmids revealed that both had suffered spontaneous deletions.The pPA102 was found to have lost 4.2 kb of DNA from 363 bp 3′ of thekanamycin resistance gene to approximately 164 bp 5′ of the start of thePA structural gene, a result consistent with the observed inactivationof the phleomycin resistance gene of pUB110. The plasmid was thenelectrotransformed into ΔSterne-1, a plasmid-free strain of B. anthracis(Infection and Immunity, 52:454-458 (1986) and transformants wereselected for kanamycin resistance. Transformants displaying a stablePA+, kanamycin resistant, (LF-, EF-, capsule-) phenotype were selected.This strain, ΔSterne-1(pPA102), was then subjected to Congo Red agarselection for mutants displaying an inability to bind the dye, acharacteristic known to correlate with an asporogenic phenotype(Worsham, submitted). The selected isolate, now designatedΔSterne-1(pPA102)CR4 was further subcultured three times to insure thata single clone was isolated. This clone has served as the seed stock forall research and development of fermentation conditions, andpurification of PA.

Materials and Methods:

Fermentation Conditions

Media: FA medium was used for all plates and liquid cultures describedhere unless otherwise specified. FA medium consisted of 33 g/l tryptone(Difco), 20 g/l yeast extract (Difco), 2 g/l L-histidine, 8 g/l Na2HPO4,7.4 g/l NaCl, 4 g/l KH2PO4 adjusted to pH 7.4 with NaOH.

Precultures: A working stock of ΔSterne-1(pPA102)CR4 was prepared fromthe seed culture by streaking cells on an FA medium plate containing 40μg/ml of kanamycin. A sweep from the confluent growth zone on plate wascultured one time in liquid FA medium supplemented with kanamycin 40μg/ml to a final O.D._(600nm) of 4.0. This culture was checked forpurity by streaking on SBA plates, and diluted into multiple vialscontaining sterile 100% glycerol to a final glycerol concentration of50% (v/v). These stocks were stored at −70° C. A single vial was removedat the start of each fermentation cycle and discarded after use. Thedefrosted cells were streaked onto FA plates containing 40 μg/mlkanamycin and incubated at least 16 hrs at 37° C. After 16 hrs theplated cells were used to inoculate 50 mls of FA medium supplementedwith 40 μg/ml kanamycin in a 250 ml baffled-Erlenmeyer flask (BellcoLaboratories). The culture was incubated at 37° C. at 200 rpm for 6 hrsor until an O.D._(600nm) of 4-6 was obtained. The cells were thensubcultured into 50 mls of FA medium in an identical flask underidentical conditions. After 6 hrs, or a culture O.D._(600nm) of 6.2-6.5,a 1.6% (v/v) inoculum was transferred to 300 mls of PA mediumsupplemented with 40 μg/ml kanamycin in a 2 liter baffled Erlenmeyer andincubated at 37° C. at 200 rpm for 7 hrs, or until a final O.D._(600nm)of 3.5-3.7 was achieved.

Fermentation conditions: The fermentations described here were carriedout using a New Brunswick Bio-Flo 3000 equipped with a 5.0 liter workingvolume glass vessel and stainless steel headplate and hemisphericalbottom cooling dish. Four liters of FA medium were added to the vessel,which had been previously completely disassembled, scrubbed in a diluteEnvirochem solution and autoclaved for 15 min after the addition of 4liters of H2O. The polarographic DO₂ probe (Ingold) and pH probes(either liquid or gel filled, Ingold) were also inserted and alladdition and sampling ports were sealed or clamped and wrapped inaluminum foil. Addition lines consisted of surgical grade autoclavableTygon tubing (Thomas Scientific) and all lines were sealed with theexception of the condenser, which was left open to permit pressurerelease, but covered with aluminum foil. The vessel was autoclaved usinga 10 min exposure time at 121° C. and removed from the autoclave as soonas sufficient cooling had occurred to allow opening of the autoclave.The vessel was then immediately connected to the fermentor unit and thecondenser line was connected to a sterile liquid trap and 0.2μ capsulefilter to avoid the introduction of contaminants during the coolingprocess. The vessel was then cooled to 37° C. using the fermentor driventemperature control and positive pressure was provided using compressedsterile filtered air. Once the vessel had cooled to 37° C. sterilefiltered kanamycin was added to a final concentration of 40 μg/ml. Theagitation was activated at 150 rpm and aeration was adjusted to 1-1.2volume/volume/min (vvm) and antifoam C (DOW), that had been diluted10-fold into H₂O and autoclaved, was added to a final concentration of200 ppm.

A preinoculation sterility check was conducted for a minimum of 16 hrsduring which time pH, agitation and temperature were continuallymonitored. After the 16 hrs required for DO₂ probe polarization, the DO₂was also monitored along with turbidity. The D₂ probe was calibratedusing an INGOLD calibration device which sets the zero value to 4 mA and100% to the oxygen tension determined by the solubility of oxygen in themedium after aeration and agitation at 37° C. The calibration andresponse of the electrode was then checked by sparging with pure N₂. Thevessel was judged to be sterile if the pH and DO₂ remained constant andno increase in turbidity was observed. It should be emphasized that theshort autoclave cycle for vessel sterilization was required to minimizecaramelization, Millard and other chemical degradation reactions whichare problematic due to the high concentrations of yeast extract andtryptone in FA medium. As an additional confirmation of sterility, 50mls was aseptically removed from the fermentor to a 250 mls Erlenmeyerand incubated at 37° C. at 200 rpm for 48 hrs with no sign of growth.Under the conditions outlined here contamination has not been observedin more than 10 fermentation cycles.

Once the sterility of the vessel had been verified, the 300 ml inoculumdescribed above was added to the vessel through the addition port of theheadplate and the initial O.D._(600nm) was recorded. A sample of theinoculum was also streaked on SBA plates and incubated for 48 hrs at 37°C. to verify inoculum purity. Using the Bio-Flo 3000, aeration wasmaintained at 75% of saturation by increasing agitation from the initial150 rpm to a maximum of 400 rpm and ultimately by supplementing the 1vvm aeration rate with pure oxygen. The mixture rate and percentages ofair and oxygen were controlled by a solenoid and algorithm developed byNev Brunswick Scientific. Both gases had a working pressure ofapproximately 10 psi.

The O.D._(600nm) dry cell weight (DCW), production of PA, DO₂, pH,agitation and temperature were monitored throughout each fermentationcycle. The O.D._(600nm) DCW and PA production analysis were carried outby manually sampling the fermentation liquor at hourly intervals using asterile sampling port. O.D._(600nm) was measured after dilution of theculture using sterile medium prepared for that fermentation. For eachO.D.600 determination, two appropriate dilutions were made and resultswere considered acceptable only when both dilutions yielded a linearresponse. DCWs were determined starting with a 2 hr point bycentrifuging 10 mls of fermentation liquor at 11,953×g for 10 min,resuspending the cell pellet in 10 mls of sterile PBS and pelleting thecells again under the same conditions. The cell pellet was resuspendedin a minimal volume of PBS and transferred quantitatively to apreweighted Eppendorf centrifuge tube and centrifuged at 14,000 rpm for5 min. Excess PBS was removed and the cell pellet was dried in aspeed-vac for 72 hrs under vacuum and a medium heat setting. A finalanalysis of the dry weight versus O.D._(600nm) revealed that therelationship between the two parameters was adequately fit with a linearfunction.

Fermentation Reproducibility: The reproducibility of the cell growthparameters, biomass and PA production in fermentations carried out withthe Bio-Flo 3000 under the conditions described above have beensummarized in Table I below. Two fermentations were carried out at 75%of the maximum dissolved oxygen concentration in a strict batch modewith no pH control or additions other than antifoam C. The variation inthe agitation rate during the first 100 min of the fermentation was theresult of the AGDO₂ (agitation DO₂) control mode chosen to maintain thedissolved oxygen tension at 75% of the maximum. Briefly, this algorithmattempts to control the oxygen tension by first altering the agitationrate until this proves insufficient, at which point the process air issupplemented with pure oxygen as needed to maintain the desired DO₂. Thetemperature was held constant at 37°+/−0.1° C. The pH was monitored, butnot regulated as an internal check on the aeration of the vessel duringthe course of the fermentation. The fact that the pH revealed a decreaseon only 0.2 pH units in the first 150 min was consistent with an aerobicculture metabolizing the limited carbohydrate supplied with the yeastextract to CO₂ and organic acids. Once the carbohydrate was exhaustedafter ca. 150 min, the bacillus switched to the utilization of aminoacids and peptides for a carbon source, which under aerobic conditionsresulted in the release of NH₄OH and the observed increased culture pH.

These fermentations were sampled on an hourly basis and allowed toproceed until no further increase in O.D._(600nm) was observed over twotime points. O.D._(600nm), DCW analysis and product measurements werecarried out for each sample as described above. Samples for PAproduction were sterile filtered followed by the addition of HEPES andthe complete protease cocktail as described under PA quantitation. Thesamples were concentrated, desalted and ultimately concentrated 80-foldprior to being analyzed using SDS-PAGE. The major band of the gelcorresponded to the 83 kDa PA product. An increasing in the intensity ofthe protein band was seen with increasing fermentation time. Study of aWestern blot of another time course of a batch fermentation wasdeveloped with polyclonal rabbit anti-PA83. Comparison revealed thatalong with increasing PA 83 kDa there was also a pronounced increase inthe abundance and form of proteolytic degradation products of PA.

TABLE 1 Summary of Aerobic ΔSterne-1(pPA102)CR4 Fermentations FinalFinal Final Yield Doubling Conc. Yield (mg Specific Time Fermentation(μg PA83/ (mg PA83/g Growth T_(D) Conditions ml PA83) DCW) Rate (min)Aerobic, Batch 51 235 8.10 0.0132 min⁻¹ 53 Aerobic, Batch 64 301 10.70.0136 min⁻¹ 51 Aerobic, Batch 45 225 7.40 0.0136 min⁻¹ 51 pH constantAerobic, 68 360 ND 0.0116 min⁻¹ 60 Fed-Batch (non- continuous) DCW = drycell weight

The data presented in Table 1 demonstrated that the PA yield on a unitvolume and biomass basis, as well as the cell growth parameters, werereproducible for the batch fermentations conducted without pH control.The final fermentation pH values of 8.57 and 8.67 after an elapsedfermentation time of ca. 8 hrs were also comparable. The effect ofprolonged exposure to these mildly alkaline conditions on cell growth,PA production and subsequent degradation was investigated by repeatingthe fermentation at a constant pH of 7.50+/−0.05 pH units. This wasaccomplished using the immersed vessel pH probe and automated additionsof 2 N HCl or 1 N NaOH. The results shown in Table 1 demonstrate thatthere was no clear effect of constant pH on any of the parametersevaluated. SDS-PAGE analysis of the fermentation time points sampled forPA production also revealed no significant differences.

The final fermentation presented in Table 1 was a noncontinuousfed-batch trial during which 1/10 volume of a 10-fold concentrate ofsterile-filtered tryptone was added after 5 hrs or an O.D._(600nm) of7.5. The result suggested that such fed-batch fermentations providepossible protocols for improvement to increase yield and decreaseproteolysis.

Harvest conditions: Fermentations were allowed to proceed until nofurther increase in O.D._(600nm) was observed. At this point, thefermentor was cooled to 10° C. and the protease inhibitorsphenylmethylsulfonyl fluoride (PMSF), 1,10-phenanthroline (OP) andethylenediamine tetraacetate (EDTA) were added to final concentrationsof 0.1, 0.05 and 2 mM, respectively. The cells were then pumped from thefermentor vessel at room temperature using an Amicon DC10L concentratorequipped with a 10-ft² 0.1 μpolysulfone hollow-fiber cartridge. Thefermentor liquor was diluted 1:1 with 25 mM diethanolamine (DEA), 50 mMNaCl, 2 mM EDTA, 0.1 mM PMSF adjusted to pH 8.9 with HCl. The filtratewas collected at an operating pressure of less than 20 psi andtransferred directly to a second Amicon DC10L equipped with two 30 kDacutoff 10-ft² wound spiral cellulosic cartridges. The filtrate wasconcentrated approximately 10-fold before being subjected todiafiltration at an operating pressure of less than 30 psi against thesame buffer. The conductivity of the retentate was monitored with anAmber Sciences conductivity meter and platinum immersion pencil-typeelectrode. The diafiltration step generally required 20 liters ofbuffer, but was considered complete only after the conductivity of theconcentrated retentate was equivalent to that of the starting buffer.

Quantitation of 83 kDa PA in crude fermentation liquor: The fermentationliquor was sampled using a sterile port at regular intervals throughoutthe fermentation process. The samples for PA determination were filteredthrough syringe type 0.2μ cellulose acetate filters, 0.1 mM PMSF, 2 mMEDTA, 50 μM OP and 20 mM HEPES pH7.3 were added and the samples werefrozen at −70° C. The samples were defrosted on ice and concentratedusing Amicon Centricon 30 concentrators at 4500×g. The samples wereconcentrated approximately 10-fold, diluted to the original volume with10 mM TRIS pH8.0, 0.1 mM PMSF, 2 mM EDTA, 0.05 pM OP and concentratedagain. The concentrated sample was desalted again using the same buffer,frozen and finally lyophilized using a Speed-Vac. The dried samples weredissolved in 25 μl of the TRIS buffer described above and diluted 1:1with a 2×SDS solubilization buffer consisting of 50 mM Na₂CO₃, 4% (w/v)SDS, 12% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol and 0.01% (w/v)Bromphenol Blue prior to heating at 95° C. for 5 min. The fermentationsamples containing varying amounts of PA 83 kDa were solubilized asdescribed above and run on a Daiichi 4-20% gradient TRIS/TRICINE gel toapproximate total yield of PA. Two hundred to 2000 ng samples ofpurified PA were solubilized in the same buffer and loaded onto the gelin constant total volume of 3 μl. Three or four appropriate dilutions ofthe fermentation samples determined from the first gel were loaded ontothe gel with the standards and electrophoresed at 100 V initially and140 V once the samples entered the separating gel and until theBromphenol Blue dye reached the bottom edge of the separating gel. Thegel was then fixed in 10% (v/v) acetic acid 20% (v/v) MeOH for 10 min,rinsed with MQ H₂O and stained with Coomassie Brilliant Blue 0.05% (w/v)in 10% (v/v) acetic acid for a minimum of 16 hrs to allow complete anduniform staining. The stained gel was then destained in 10% (v/v) aceticacid until the background contained no visible residual dye. The gel wasthen scanned on a laser densitometer (LKB, Ultrascan XL LaserDensitometer). Representative portions of the gel without protein wererandomly chosen and scanned to determine background absorption for anaccurate baseline. The region to be scanned for each lane containing PAwas then visually aligned to insure that the entire protein peak andadequate baseline were included in each scan. The scans were completedand the integration values were determined using the LKB preprogrammedGaussian algorithm and later were confirmed by cutting out individualpeaks and manually integrating based on peak weight. The resultingintegration values were plotted using Sigmaplot (Jandel). Linearregression of the results revealed typical r values of 0.992-0.996. Thelinear standard curve was then used to quantitate the amount of 83 kDaPA in the various fermentation samples based on the same integrationmethods.

Purification: The exact volume and conductivity of the PA in DEA bufferwas determined and solid KCl was added to the solution to a finalconcentration of 30 mM and conductivity of 10-11 mmhos/cm. The PA waspumped with a peristaltic pump through a monoQ column prepared bycollecting 100 mls of hydrated Bio-Rad Macro Prep 50Q on a sinteredglass filter and washing sequentially with 1 liter of 25 mM DEA, 50 mMNaCl, 1 mM EDTA, 50 μM OP and 0.1 mM PMSF pH8.9 and 1 liter of the samebuffer with 30 mM KCl added. The conductivity (10-11 mmhos/cm) and pH of8.9 of the eluate from the Macro Prep 50Q after the second wash werecomparable to that of the PA solution after addition of KCl. The MacroPrep 50Q resin was then degassed and slurry packed into a Pharmacia Kcolumn with a Rainin Rabbit-Plus peristaltic pump at 48 rpm and a flowrate of 15 mls/min. The final column volume was (5×5 cm) 98 mls. The PAsolution was pumped through the Macro Prep 50Q column at a rate of 10mls/min and the eluate was collected until all of the PA sample volumewas loaded and the column washed with an additional 100 mls of DEA/KClbuffer. The eluate containing unbound PA was concentrated anddiafiltered using an 1-ft² 30 kDa cutoff cellulosic Amicon wound spiralcartridge at an operating pressure of 20 psi.

The final concentrate (ca. 400 mls, 6-7 mmhos/cm) was passed through a0.2μ cellulose acetate filter. The filtered PA was loaded onto a PorosIIQ perfusion chromatography column using a quaternary Waters 600E HPLCpump. The column was prepared by hydrating seven grams of the Poros IIQperfusion resin in twice the packed bed volume of 2% (w/v) NaCl. Aftersettling the resin was resuspended in six times the packed bed volume of25 mM DEA pH 8.9, 50 mM NaCl, 7.5% (v/v) ethylene glycol and allowed tosettle overnight at room temperature. The resin was then resuspended inthree times the packed bed volume and finally in one and one-half timesthe final volume before the slurry was extensively degassed using avacuum pump (vacuum unknown). The entire degassed slurry was thentransferred to a Waters AP 20×100 mm glass HPLC column and the columnwas packed in one step using the Waters 600E pumps at a flow rate of 20mls/min and a backpressure of 650 psi at room temperature. The columnseparation efficiency was then tested at a flow rate of 10 mls/min usinga linear 1 M NaCl gradient and ovalbumin 5 mg/ml (Sigma) and bovineserum albumin 10 mg/ml (Sigma) in DEA as buffer as standard proteins.Approximately 100 mls of PA (ca. 20-30 mg PA) cooled to 4-6° C. wasapplied to the column and followed with a 20 min wash in the startingbuffer at room temperature to elute unbound material. The column wasthen developed with a linear gradient to 30% of the 1 M NaCl DEA elutionbuffer. The purified PA was found to elute between 10-15%, while thesmaller molecular weight proteolytic breakdown products eluted as ashoulder or partially resolved peak at 16-20% of the elution buffer. Theresolution of the two peaks was found to be a function of content of PAproteolytic degradation products. The eluant was monitored at 280 nm andpeak fractions were collected by manual triggering of an ISCO fractioncollector. Samples of the peak fractions were diluted into 5-10 volumesof TRIS pH8.0, 0.1 mM PMSF, 50 AM OP, 1 mM EDTA buffer and concentratedusing Amicon Centricon 30 concentrators at 4500×g at 4° C. toapproximately the initial sample volume. An equal volume of SDS-PAGEsolubilization buffer was added to the sample immediately prior toheating at 95° C. for 5 min. Purity was assessed from 8-25% SDS-PAGEPHAST gels (Pharmacia) and fractions with the highest purity werecombined and dialyzed against 40-50 volumes of 25 mM DEA pH8.9, 50 mMNaCl, 0.1 mM PMSF and 2 mM EDTA at 4° C. for at least 16 hrs. Fractionsjudged empirically to be less than 95% pure were rechromatographed underthe same conditions and purity of the fractions was reassessed asdescribed above. All fractions of greater than 95% purity wereultimately combined, aliquoted and frozen at −70° C. subsequent todetermination of the total PA concentration.

Analysis and characterization of purified 83 kDa PA: Purified PA wasquantitated by measuring UV-absorption at 280 nm using the relationshipof 1 A_(280nm) in a 1 cm pathlength cuvette is equals 1 mg PA/ml(Leppla, 1988). Results obtained in this manner were confirmed using theBio-Rad Bradford protein assay under conditions suggested by themanufacturer. PA purity was assessed using SDS-PAGE under conditionsdescribed above. Capillary electrophoresis analytical assays have alsoproven promising in the assessment of PA purity and amounts of residualprotease inhibitors in final product. Feasibility studies using a 47cm×50 pm uncoated silica capillary and borate/SDS/acetonitrile bufferrevealed an excellent separation of the protein from residual proteaseinhibitors. Quantitation of both protein and inhibitors has also provenpossible, but the technique remains limited by the relatively highlimits of detection (1 mM EDTA, 0.1 mM PMSF, and 0.05 mM OP) undercurrent conditions. Automated N-terminal sequencing was carried out withpurified PA using an Applied Biosystems 470A sequenator after desaltingover Bio-Rad PD10 columns equilibrated with 5 mM NaCl and 1 mM CaCl₂. Aunique N-terminal sequence was found and the first six residues of thesequence were identical to PA from the endogenous plasmid pXO1 harboredby the USAMRIID B. anthracis Sterne strain. In addition, the sequencecorresponded exactly with the published DNA derived protein sequence(Welkos et al.). Native gel electrophoresis under non-denaturingconditions revealed that PA purified from ΔSterne-1(pPA102)CR4 alsoexhibited the microheterogeneity noted previously for PA produced by theSterne strain. Cytotoxicity assays of the product using the macrophagelysis assay (Friedlander et al.) revealed that the titration curve ofbiological activity for PA from ΔSterne-1(pPA102)CR4 wasindistinguishable from that generated for PA from the Sterne strain.

Evaluation of ΔSterne-1(pPA102)CR4:

EXAMPLE 1

B. Anthracis ΔSterne-1(pPA102)CR4 was compared with its parentspore-forming strain B. anthracis ΔSterne-1(pPA102). Both organisms wereplated onto sheep blood agar (a preferred medium for promoting bacterialspore production) and grown at 37° C. for 1 day, after which thetemperature was lowered to 25° C. for 4 days. The two strains were alsogrown in liquid Leighton-Doi medium, which is designed to promote sporeproduction, for 1 day at 37° C. followed by 4 days growth at 25° C.Growth from both agar and broth cultures were examined under phasecontrast microscopy for the presence of spores. Growth from all fourcultures were then resuspended in phosphate buffered saline to aconcentration of about 10⁹ colony-forming units (CFU) per ml. All fourcultures were then heat shocked at 64° C. for 60 minutes to killvegetative cells. Aliquots of 0.1 ml of the heat shocked material wasthen plated out onto sheep blood agar and incubated at 37° C. for 2days.

Results:

B. anthracis ΔSterne-1(pPA102): Spores were seen under microscopicexamination of material from both the sheep blood agar cultures and theLeighton-Doi medium cultures. On sheep blood agar plates containing heatshocked culture material from both sheep blood agar cultures andLeighton-Doi medium cultures, there was confluent growth. The dataclearly indicate that B. anthracis ΔSterne-1(pPA102) forms spores.

B. anthracis ΔSterne-1(pPA102) CR4: No spores were seen undermicroscopic examination of material from both the sheep blood agarcultures and the Leighton-Doi medium cultures. On sheep blood agarplates containing heat shocked cultures, there was no growth whatsoever.The data clearly indicate the B. anthracis ΔSterne-1(PPA102) CR4, whichhas been deposited in the American Type Culture Collection and has beenassigned ATCC designation 69714, does not form spores. The deposit atthe American Type Culture Collection located at 12301 Parklawn Drive,Rockville, Md. 20852, USA was made on Nov. 16, 1994.

EXAMPLE 2

B. anthracis ΔSterne-1(pPA102)CR4 was grown in an FA medium fermentorculture. No spores were seen upon phase contract microscopicexamination. Only medium-length and long chains of bacilli were seen.Dilution plate counts on the culture determined that the culturecontained 1.86×10⁹ CFU per ml. Three ml of culture was heat shocked at60° C. for 60 minutes, then 0.2 ml was plated onto each of 5 plates ofTryptic soy agar. After incubation for 2 days at 37° C., no colonieswere seen on the agar plates, indicating that spore production in thefermentor was less than 1 per 1.86×10⁹ CFU. On two other fermentationruns with this strain, similar results were obtained. No revertants tothe parent spore-forming phenotype were observed.

The above process using an FA medium fermentor culture was repeatedusing the parent strain B. anthracis ΔSterne-1(pPA102). Growth on thetryptic soy agar after heat shock resulted in a total of 1000 totalcolonies, indicating that the parent strain B. anthracisΔSterne-1(pPA102) had about 1000 spores per ml in the FA medium, or 1spore per 106 CFU in the non-heat shocked medium.

EXAMPLE 3

Protective antigen (PA) was prepared in accord with the teachings underMaterials and Methods as described above. The purified PA of B.anthracis ΔStern-1(pPA102)CR4 was mixed in different buffers (phosphatebuffered saline, HEPES, Tris, glycyl glycine (GG), sodium citrate, forexample) and combined with monophosphoryl lipid A (MPL), Squalene, Tween80 and lecithin. The mixture was then lyophilized. At 0 and 4 weeks,vials of lyophilized MPL/PA/emulsion were reconstituted in phosphatebuffered saline (PBS) and injected in 0.5 ml doses containing 50 μg ofPA per dose. At 10 weeks, the guinea pigs were aerosol challenged withapproximately 36 medial lethal doses of virulent Bacillus anthracisspores of the Ames strain. The following data shows status two weeksafter the challenge.

Vaccine S/T* % Anti-PA** PA in PBS (+ MPL emulsion) 10/12 83 29,427 PAin GG (+ MPL emulsion) 14/16 88 23,713 PA in Tris (+ MPL emulsion) 15/1694 27,384 PA in HEPES (+ MPL emulsion) 15/15 100  25,482 PA in Citrate(+ MPL emulsion) 16/16 100  31,622 PBS 0/4  0   <10 *Survived/Total, day14 post-challenge **Prechallenge serum titers to PA were determined byenzyme linked immunosorbent assay. The geometric mean reciprocal titerswere calculated for each group and are expressed in this table.

What is claimed is:
 1. A method of making a vaccine comprising:incorporating a protective antigen produced by recombinant asporogenicB. anthracis with a pharmaceutically acceptable carrier, wherein saidrecombinant asporogenic B. anthracis was isolated from aΔSterne-1(pPA102) strain of bacteria and said recombinant asporogenic B.anthracis does not have the ability to bind a dye when grown on CongoRed Agar.
 2. The method of claim 1, wherein the recombinant asporogenicB. anthracis is B. Anthracis ΔSterne-1(pPA 102)CR4.
 3. The method ofclaim 1, wherein the vaccine is in the form of a suspension.
 4. Themethod of claim 1 wherein the vaccine is in the form of bufferedsuspension.
 5. The method of claim 1 wherein said carrier is anadjuvant.